11-microbiological hydroxylation of steroids in presence of dimethylsulfoxide



United States Patent 10,791 12 Claims. (Cl. 195-51) The presentinvention relates to microbial transformations of steroids.

More particularly, the present invention relates to a novel process forconducting certain microbiological oxidative transformations of steroidsby known strains of microorganisms, or by the enzymes they produce, inthe presence of dimethylsulfoxide or derivatives thereof.

While the present invention has application to a wide variety ofmicrobiological transformations, it may be more readily understood byreference to the commercially important ll-hydroxylation of steroids.

The 11-hydroxylation of steroids using various strains of fungi andbacteria is a well-known process which has been commercially used for anumber of years. For example, there has been disclosed the use of fungiof the order Mucorales for the ll-hydroxylation of various steroids. Ingeneral, some of these fungi introduced an llot-hydroxyl group andcertain others of the order Mucorales, such as Cumzinghamellablakesleeana, bainieri, elegans and echinulata, introduce anllp-hydroxyl group. Various patents and publications have taught thatother families and species of fungi and bacteria also possess theability to introduce the ll-hydroxyl group, as for example Curvularia,Streptomyces, etc.

In general, the yields obtained by the processes previously set forthhave not been quantitative and rarely exceeded 50% of thell-hydroxylated steroid based on starting material, although in someinstances, using certain specific steroids and cultures, the yields havebeen somewhat greater than this amount.

In accordance with the present invention the surprising discovery hasbeen made that the addition of dimethylsulfoxide to the fermentationmedium produces a significant beneficial effect on the ll-hydroxylationprocess. While not intending to be bound by any theory herein advancedin explanation of possible mechanisms, it is believed that the observedbeneficial effect is a manifestation of changes in the nature oftransport across cell membranes of either or both the enzyme andsteroid, of changes in the activity of the various enzymes present inthe system, of changes in the growth capabilities of contam-inantmicroorganisms, and/or of changes in the extraction of the steroidalmaterial from the lnycelium. These possibilities are not exhaustive norare they mutually exclusive, and quite possibly more than one mechanismis involved.

Further typifying the practice of this invention, it has been found thatif an amount from about 1% to about 50%, preferably less than 20% andideally up to about of dimethylsulfoxide is introduced into afermentation broth of an oxidative microorganism, such as for examplethe 11-hydroxylating organisms Cunninghamella blakesleeana, Curvularfialunata, or any of the many others known to the art, conversion of thesteroid upon incubation for periods from about 4 to about 48 hours iseffected with a greater yield of and with a greater total recovery ofthe ll-hydroxy steroid as well as with a concomitant reduction in theamountof undesired side products. It will be recognized, of course, thatthe optimum amount of dimethylsulfoxide will vary not only with theparticular microorganism employed but also with the particularconditions of incubation, including rate of aeration, broth composition,size and type of fermentation equipment, and the like. Thus, forexample, while the op timum amount of dimethylsulfoxide for thelip-hydroxylation of a preferred steroid, 6ot-fluoro-16,17-acetonidecompound S, with the microorganism, Cunninghamella blakesleeana,A.T.C.C. NO. 868812, is from about 7% to about 10% by volume based onthe total initial volume of the fermentation medium, this amount willnot necessarily apply to different transformations, different organismsor different steroidal starting materials, eg compound S.

It is expected that in the environment of the fermentation medium, thedimethylsulfoxide may be itself converted to other derivatives, and thatone or more of these derivatives are responsible for the beneficialeffects observed. Among such possibilities are dimethylsulfide,dimethylsulfone, methylsulfenic acid, methylsulfinic acid and/ ormethylsulfonic acid. Dimethylsulfoxide is, however, quite satisfactoryin terms of cost and ease of handling, and such derivatives thereof areconsidered as merely possible substitutes.

Essentially, any steroid compound containing from 18 to 27 carbon atomsinclusive, which is unsubstituted at C-1l, may be used as startingmaterial for this aspect of the process of the present invention. Thus,the starting materials belong especially to the androstane, estrane,pregnane, cholestane and sapogenin series. They may possess a keto orhydroxyl group at C3i or may be unsubstituted at said position, and maybe saturated or unsaturated. Double bonds may be present at C-1, C-4,C-5, C-6, 0-16, etc. or combinations of the same. A great variety ofsubstituents may be also present in one or several positions of thesteroid molecule, and particularly halogen atoms at C-2, 6, 16 and/or21; alkyl groups at C-2, 4, 6, 16, 17, etc., alkylidenedioxy groups atC--t, etc. Other substituents may be also present at C-1, 2, 4, 6, 7,12, 14, 15, 16, 17, 18,. 19, 20 and 21, e.g. a keto group at 0-20.

Suitable starting materials thus include:

testosterone,

l9-nor-testosterone,

l7a-methyltestosterone,

17ot-vinyltestosterone, l7a-ethyny1-m nor-testosterone,dihydroallotestosterone,2-hydroxymethylene-17a-methyldihydroallotestosterone, A -androstene-3B,17,8-diol, A -andr0stene-3,l7-dione,

progesterone,

l7a-hydroxyprogesterone, l7a-acetoxyprogesterone,

pregnenolone,

allopregnenolone, 6a-methyl-l7ot-hydroxyprogesterone,6a-fluoro-17a-hydroxyprogesterone,6chloro-6-dehydro-l7a-hydroxyprogesterone, 2 Lfluoroprogesterone,

3-desoxyprogesterone,

19-nor-progesterone,

desoxycorticosterone, M-pregnene-l7a,2l-diol-3,20 dione (compound6a-methyl S,

6a-chloro S,

16a-methyl S,

IGB-methyl S,

16a-hydroxy S,

16OL,170L'&CetOI1ide of 16a-hydroxy S, 6a-fluoro-l6u-methyl S,

16a,17a-acetonide of 6a-fluoro-16whydroxy S, Apregnene-3,B,17a,21-triol-2O-one, 6,l6a-dimethy1 S,allopregnane-3fl,l7a,2l-triol-20-one,allopregnane-l7a,2l-diol-3,20-dione,16a-methylallopregnane-17u,2l-diol-3,20-di0ne, l-dehydro S,

6-dehydro S,

l-dehydro- 1 6a-methyl S,

1,6-bisdehydro S, 6-fluoro-16ot-methyl-1-dehydro S,

estrone,

estradiol,

cholesterol,

choleste'none,

cholanic acids,

diosgenin,

tigogenin, etc.

The microorganisms used in this process include those fungi and bacteriadescribed in the literature as effecting 1 1(1- and l lfi hydroxylation.

The fungi used generally belong to the classes Phycomyceteae,Ascomyceteae, Basidiomyceteae and Fungi Imperfecti.

The Phycomycetes more commonly used for ll-hydroxylation belong to theorder Mucorales, family Mucoraceae, especially those species belongingto the genus Absidia or Rhizopus such as for example Absidia glauca,Rhizopus nigricans and Rhizopus arrhizus, or to the familyChoanephoraceae, particularly the genus Cunninghamella, especiallyCunninghamella blakesleeana, Cunninghamella bainieri, Cunninghamellaelegans and Cunninghamella echinulata.

The Ascomycetes used for this type of reaction belong to the ordersHypocreales, Sphaeriales and Eurotiales, such as for example Gibercllafujikuroi, Neurospora sitaphilu and Eurotium chevalieri, respectively.

The Basidiomycetes used for ll-hydroxylation belong particularly to theorder Agaricales, family Agaricaceae, of the genera Psilocybe,Stropharia and Conobybe, and specifically the species Panaeoluscompanulatus and Psilocybe caerulescens var. Ma-zatecorum.

Examples of suitable Fungi Imperfecti used in our process are:Curvularia lunala, F usarium monilifarme and other Fusarium species,Stachylidium bicola, Helminthosporium sativum, Cephalotecium roseum,Pestalotia feet/ans, Dactylium demlroides, A spergillus ochraceus,Aspergillus niger and other Aspergillus species, Penicilliu'mehrysogenum, Penicillium notatum, Penicillium nigricans, Penicillium roqueforti and other Penicillium species, Spondylocladium australe,Arthrobotrys superba, Coniothyrium species, Pycnosporium species,Rhodoseptoria species, Trichothecium roseum, Stachylidium theobromae,Strachylidium bicola, and Epicoccum species.

Other microorganisms belonging to the bacteria (order Actinomycetales)and specially Streptomyces species, such as Streplomyces fradiae, mayalso be used in this process.

The culture media will contain mainly a source of carbon, a source ofnitrogen, cofactors and minerals. Examples of carbon sources includecarbohydrates such as glucose, maltose, mannose, dextrose, lactose,sucrose, galactose, molasses and the like, polyalcohols such asglycerol, manitol and the like, starches, etc.

Adequate sources of organic nitrogen are vegetable or animal proteinssuch as soybean meal, corn steep liquor, corn-meal, lactalbumin,caseine, peptones, amino acids or commercial products such as Phytone(enzymatic digest of soya meal), Casitone or Edamine (lactalbuminhydrolyzates) Micophil (soybean protein), Nutrient L-l (lactalbuminhydrolyzate), NZ-amine (pancreatic hydrolyzate of c-aseine), and othersimilar materials. There may be also used as sources of nitrogen,nitrates or ammonium salts such as sodium or potassium nitrate, ammoniumnitrate, ammonium sulfate, ammonium hydroxide solutions urea, etc.

The mineral constituents are present in the form of salts, speciallychlorides, phosphates and sulfates of sodium, potassium, iron,manganese, etc.

Yeast extract, vitamins, decoctions of potato, etc. may be mentioned ascofactors.

In general, the liquid culture media used in our process are those usedin the known process, and vary in accordance with the oxygenatingmicroorganism.

An antifoaming agent, such as for example silicone, glyceride oils andwaxes, soybean oil, castor oil, sulfonated oils, mineral oils, and thelike may be added. The pH of the culture media is adjusted at theoptimum pH for the growing of each microorganism.

In practicing the process of the present invention, a mycelial growth ofthe oxygenating microorganism is first obtained through incubation of aculture of the same in the appropriate liquid medium for a period oftime of between 6 to 48 hours, preferably for 24 hours. There is thencarefully added the desired amount of dimethylsulfoxide, taking carethat the temperature of the medium does not exceed about 28 C. To thismixture is then added the steroid used as substrate, either in solidform or dissolved in the minimum amount of a solvent such as ethanol,dioxane, acetone, etc. Alternatively, the steroid to be hydroxylated maybe dissolved in a small amount of the total dimethylsulfoxide to be usedin the medium. The amount of steroid utilized is essentially the same asheretofore employed, keeping in mind such things as inhibition. Maximization of the amount of steroid can often be realized by adding thesteroid at the same rate it is disappearing, i.e., being converted. Oncesteroid is added, incubation is resumed for a period of time of between4 to 48 hours, depending on the substrate. The point of optimumconversion may be determined by periodically talking aliquots which maybe analyzed by paper chromatography.

When the reaction is complete, the mixture is extracted with severalvolumes of an organic, water-immiscible solvent such as ether, methylenedichloride, ethyl acetate and the like, using preferably methylenedichloride, and the organic extract is washed several times with waterto eliminate the dimethylsulfoxide, re-extracting the combined washingswith methylene dichloride to avoid the loss of steroid. The combinedextract is dried, evaporated to dryness and purified by the usualmethods.

This process may be also effected by using instead of a culture of themicroorganism, a solution of the enzymes produced thereby, or with asuspension of spores. While the process of the present invention hasbeen typified above in its application to ll-hydroxylation, the value ofdimethylsulfoxide in these applications extends to other microbiologicaloxidative conversions. Among these may be mentioned the microbiologicalside chain degradation of sterols such as cholesterol,19-hydroxy-cholesterol, 3- acetate and the like with microorganisms ofthe order Schizomycetes, family Actinomycetales, most notably Nocara'iarestrictus, A.T.C.C. No. 14887, and certain species of Mycobacteriumincluding Mycobacterium flavescens, Mycobacterium phlei, A.T.C.C. No.10142, Mycobaclerium acapulcensis, A.T.C.C. No. 15414, and the like.

The following examples serve to typify the nature of this invention, butbeing presented solely for the purpose of illustration, they are notintended to limit it.

EXAMPLE 1 There was prepared a culture of Cunninghamella blakesleecma,A.T.T.C. 86881), in five l-liter Erlenmeyer flasks containing 200 cc.each of a culture medium of the following composition: Grams Soybeanmeal 5 Yeast extract (Difco) ,5 K HPO 5 NaCl 5 Cerelose (commercialglucose) 20 Distilled water to complete 1 liter.

The pH of this culture medium was adjusted to 7.2.

After 24 hours of growing at 2830 C. under aeration and stirring (250rpm), the content of the flasks was combined and added to a stainlesssteel fermentor of 14 liters capacity containing 4 liters of the sameculture medium and g. of Silicone A as an antifoaming agent. There werethen carefully added 500 cc. of dimethylsulfoxide (10% by volume), whilemaintaining the temperature below 28 C. and 500 mg. of6a-fiL1OI0-l6u,17ocisopropylidenedioxy-M-pregnene-Z1 ol 3,20 dionedissolved in 50 cc. of 95% ethanol, and the incubation was resumed atthe same temperature under stirring (273 rpm.) and aeration (lvol./lt./min.) for 12 hours further. At the end of this time the productwas extracted three times with methylene chloride and the organicextract was washed several times with water to eliminate thedimethylsulfoxide, re-extracting the washings to avoid loss of steroid.The combined extracts were dried over anhydrous sodium sulfate andevaporated to dryness under reduced pressure. The solid residue waspurified by chromatography on silica gel-Celite diatomaceous earth, thusyielding 60 fluoro 16a,17a-isopropylidenedioxy-A-pregnene-l15,21-diol-3,20-dione (acetonide of6a-fluoro-l6uhydroxy-hydrocortisone) in pure form, identical to anauthentic sample, in 78% yield.

EXAMPLE 2 The preceding example was repeated but the dimethylsulfoxidewas omitted. In this case there was obtained the acetonide of6ot-fl1101'0-16oz-hYdIOXY-hYdlOCOItiSOIlfi in 30% yield.

EXAMPLE 3 In the method of Example 1 the incubation time was reduced to6 hours to produce the acetonide of 6u-fluoro-16a-hydroxy-hydrocortisone in 68%yie1d.

EXAMPLE 4 In the method of Example 1 the incubation time was reduced to8 hours, thus yielding the llfl-hydroxylated compound in 72% yield.

EXAMPLE 5 Example 1 was repeated but by volume of dimethylsulfoxide wasadded to produce the acetonide of 6ix-fluoro-16u-hydroxy-hydrocortisonein 50% yield.

In another experiment there were used equal amounts of the culturemedium and dimethylsulfoxide, thus obtaining a 40% yield of thehydroxylated product.

EXAMPLE 6 In the method of Example 1 the incubation time was extended to12 hours to produce the acetonide of 60afluoro-16a-hydroxyhydrocortisonein 70% yield.

EXAMPLE 7 I II Com ound S Hydrocortisone. Acet nide of16a-hydr0xy-S"Acetoinide of lfia-hydroxyhydrocor isone.

fia-ehlorohydroeortisone.

fia-methylhydrocortisone.

lfia-methylhydrocortisone.

IGB-methylhydrocortisone.

fia-fluoro-lGa-methyihydrocortisone.6a-flu0ro-1fifi-methylhydrocortisone. lIB-hydroxyprogesterone.

I II

17u-hydroxyprogesterone 11B,17a-dihydroxyprogesterone.6a-methyl-l7a-acetoxyprogestoronefia-methyl-llehydroxy'l7a-acetoxyprogesterone. 6-ehloro-A -pregnadien-l7a-0l- 6-eh10ro-A --pregnadiene-11B,17a-

3,20-dione acetate.

dione. 3,20-d10ne. 6a-lluoro-A -pregna dione-1 701,21- fia-fluoro-Apregnadiene-ll 13,1701,

d1ol3,20-d1one. 21-tr1ol-3,20-dione.

6a-fluoro-16a-methyLA -pregnadione-17a,21-diol-3,20-dione.

Testosterone llfl-hydroxytestosterone. 17a-methyltestosterone17a-rnothyl-1lfl-hydroxytestosterone. 17a-ethyny1-19-nortestosterone17a-ethynyl-1lfl-hydroxy-ltq'nortestosterone. Estronellfl-hydroxyestrone. Cholestenone llfl-hydroxyoholestenone. D osgen nllfi-hydroxydiosgenin. Tlgogenm llfl-hydroxytigogenin.

EXAMPLE 8 6oz fiuoro 160;,17! isopropylidenedioxy-M-pregnen-2l-ol-3,20-dione is subjected to llB-hydroxylation by incubation with aculture of Clmningham ella blakesleeana in accordance with the procedureof Example 1 utilizing, however, 250 cc. of dimethylsulfoxide (5% byvolume). Upon completion of the procedure as therein described,oa-fiuoro 160,17u-isopropylidenedioxy-A -pregnene-11,3,21-diol-3,20-dione is obtained in approximately 70% yield.

EXAMPLE 10 A culture of Rhizopus nigricans, A.T.C.C. 6227b, was preparedby inoculating an aqueous medium which contained 2% of peptone and 5% ofcorn syrup with a vegetating growth of the above culture in the same medium and stirring at 28 C. with aeration for 24 hours.

To 340 cc. of this culture there were then slowly added 30 cc. ofdimethylsulfoxide while maintaining the temperature around 28 C., and100 mg. of progesterone dissolved in 4 cc. of dimethylsulfoxide, and themixture was stirred with aeration at 28 C. for 24 hours further. Theproduct of this incubation was extracted several times with methylenechloride, the extract was washed with water, dried over anhydrous sodiumsulfate and concentrated to a small volume under reduced pressure.

The concentrated extracts were adsorbed on a column prepared with 20 g.of silica gel and 20 g. of Celite diatomaceous earth washed previouslywith methylene chloride. Elution with a mixture of methylene chloride:acetone :20 and crystallization gave lla-hydroxy progesterone in 75%yield.

EXAMPLE 11 A strain of Curvularia lunata, A.T.C.C. 13935, was grown in aSabouraud glucose-agar medium (Difco). The growth obtained afterincubating for a week at 25 C. was suspended in 5 cc. of sterile water.This suspension was divided in five portions of 1 cc. each, which wereemployed for inoculating five Erlenmeyer flasks of 250 cc. capacitycontaining 50 cc. each of a culture medium of the following composition:

Distilled water to comple 1 liter.

The cultures were incubated under rotatory stirring for 72 hours at 25C. The growth was homogenized for 1 minute in a Waring Blendor, and 2cc. portions of the suspension thus obtained were employed forinoculating 100 Erlenmeyer flasks containing the same amount of themedium described above. The mixtures were incubated for 24 hours underrotatory stirring at 25 C. and 280 r.p.m. To each flask there were added5 cc. of dimethylsulfoxide and 50 mg. of compound S dissolved in 2 cc.of 95% ethanol, and the incubation was continued under the sameconditions for 24 hours. The contents of the flasks were combined andextracted with four portions of methylene chloride. The combined extractwas Washed well with water to eliminate the dimethylsulfoxide, driedover anhydrous sodium sulfate and concentrated at low temperature to avolume of 25 cc. This solution was adsorbed on 4 g. of silica gel andeluted with methylene chlloiridemther (9:1) to produce hydrocortisone in75% we EXAMPLE 12 A culture of Psz'locybe caerulescens var. Mazatecorum,(Heim), A.T.C.C. 13964, was maintained by serial trans ference every twoweeks, in a mycophyl-agar or maltagar medium, incubating at atemperature of 2528 C.

The growth obtained in an inclined agar tube was suspended in cc. ofsterile water, and 2 cc. of this suspension were used to inoculate anErlenmeyer flask containing 200 cc. of the following culture medium:

Phytone grams 2 Dextrose do 2 Water to cc 200 The cultures wereincubated under rotatory stirring (200 r.p.m.) at 2528 C. for threedays. The rnycelium thus obtained was dispersed using a blender, and cc.of the microbial suspension thus obtained was inoculated to each of tenl-liter Erlenmeyer flasks containing 200 cc. each of the same culturemedium and then incubated for 24 hours further. To each flask there werethen slowly added 10 cc. of dimethylsulfoxide.

To each flask there was added 50 mg. of the 3,21-diacetate of A-pregnene-3B,17u,21-triol-20-one, and the stirring under aeration wascontinued for 48 hours further at the same temperature. The content ofthe flasks was combined and extracted several times with methylenechloride, the extract was washed with water, dried over anhydrous sodiumsulfate and evaporated to dryness under reduced pressure. The residuewas adsorbed in a column charged with 12 g. of silica gel and 12 g. ofCelite diatomaceous earth, thus obtaining A -pregnene-11a,17 t,-21-triol-3,20-dione, M.P. 217--219 C. identical with an authentic sampleof ll-epi F.

EXAMPLE 13 The vegetative growth of Giberella fujikuroi (Fusariummoniliforme), A.T.C.C. 11161, obtained after one weeks incubation at 25C. in an inclined test tube containing a potato dextrose-agar medium,was suspended in 10 cc. of sterile water. One cc. of this suspension wasthen used to inoculate ten l-liter Erlenmeyer flasks, each containing200 cc. of Czapeks solution supplemented with 0.05% of yeast extract.The flasks were stirred in the presence of air under submergedconditions (rotatory shakers operated at 150 r.p.m.) for 18 to 21 hoursto obtain an abundant growth of the microorganism. To each flask therewas then added 15 cc. of dimethylsulfoxide, maintainin the temperatureat 25-28 C., and 50 mg. of 6ot-fluoro-16u,- l7a-isopropylidenedioxy-Apregnen 21 ol 3,20-dione, and the incubation was resumed for 18 hoursfurther un-' der the same conditions. Following this incubation period,the contents of the flasks were combined and then extracted severaltimes with methylene chloride, and the extract was washed well withwater, dried over anhydrous sodium sulfate and evaporated to drynessunder reduced pressure. The residue was dissolved in methylene chloride,adsorbed in a column charged with 15 g. of silica gel and 15 g. ofCelite diatomaceous earth. The fractions eluted from the column withether and etherzacetone (:10) were found to contain 400 mg. of6ot-fluoro-16a,- 17a-isopropylidenedioxy-A -pregnene 1104,21 diol-3,20-dione identical to an authentic sample.

What is claimed is:

1. In the process for producing ll-hydroxylated steroids by incubatingthe corresponding ll-desoxy steroid with a microorganism capable ofeffecting said hydroxylation or with the enzymes produced thereby, theimprovement comprising conducting said incubation in the presence ofdimethylsulfoxide.

2. A process of claim 1 wherein the steroid is a member of theandrostane, pregnane, estrane, cholestane or sapogenin series, and theincubation is in a liquid medium for a period of from 4 to 48 hours.

3. A process of claim 1 wherein the amount of dimethylsulfoxide used isfrom about 1% to about 50% of the total volume of the culture medium.

4. A process of claim 3 wherein the amount of dimethylsulfoxide used isup to about 10% of the total volume of the culture medium.

5. A process in accordance with claim 4- wherein the oxygenatingmicroorganism belongs to the genus Cunninghamella and the steroid is ofthe pregnane series.

6. A process in accordance with claim 4 wherein the oxygenatingmicroorganism belongs to the genus Rhizopus and the steroid is of thepregnane series.

7. A process in accordance with claim 4 wherein the oxygenatingmicroorganism is Curvularia ltmata and the steroid is of the p-regnaneseries.

8. A process of claim 5 wherein the oxygenating microorganism isCunninghamella blakesleeana.

9. A process of claim 5 wherein the oxygenating microorganism isCunninghamella bainieri.

10. The process of claim 4 wherein the oxygenating microorganism isFusarium mom'liforme and the steroid is of the pregnane series.

11. A process for producing 6ot-fluoro-16a,17ot-isopropyl-idenedioxy-A-pregnene-11 8,21 diol 3,20-dione, which comprises incubating6a-fluoro-16u,17ot-isopropylidenedioxy-N-pregnene-Zl-ol-3,20-dione witha culture of Cunninghamella blakesleeana, A.T.C.C. 868812, in a liquidculture medium containing dimethylsulfoxide.

12. The process of claim 11 wherein the liquid culture medium containsup to about 10% of dimethylsulfoxide.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, PrimaryExaminer.

1. IN THE PROCESS FOR PRODUCING 11-HYDROXYLATED STEROIDS BY INCUBATINGTHE CORRESPONDING 11-DESOXY STEROID WITH A MICROORGANISM CAPABLE OFEFFECTING SAID HYDROXYLATION OR WITH THE ENZYMES PRODUCED THEREBY, THEIMPROVEMENT COMPRISING CONDUCTING SAID INCUBATION IN THE PRESENCE OFDIMETHYLSULFOXIDE.